The objective of this project is to better understand the mechanisms which support the function of the cholinergic synapse. Experiments will be designed and implemented to determine if the high affinity choline co-transporter (ChCoT), like its relative the GLUT-4, is translocated in carrier (cargo) vesicles and recruited to the terminal membrane by an Exocytotic process which involves a specific syntaxin, a SNAP-25 homolog, and other SNARE proteins. Efforts will be made to immunoisolate the vesicle and partially characterize vesicle associated proteins involved in this process. In order to accomplish this goal, a ChCoT -specific antibody will be developed and used to immunoisolate the ChCoT. In addition, the antibody will be used to distinguish the ChCoT from other neurotransmitter vesicles. Furthermore, proteins associated with the ChCoT vesicle, such as ChCoT- and SNARE proteins, will be characterized. Lastly, the syntaxin requirements necessary for activity dependent upregulation of the ChCoT will be established. Successful completion of this project will have significant implications for studying the molecular mechanisms undergirding membrane trafficking of the choline co-transporter, as well as furthering our understanding of nervous system pathologies in which cholinergic pathways are implicated.